Development of Triploid Callus of Hevea brasiliensis Using Endosperm

Abstract

Aim: To generate a pathway for development of Hevea triploids using endosperm tissue as an explant. Study Design: Standardization and optimisation of various parameters for isolation and culture of endosperm tissue and protoplast. Completely randomized design for data from different treatment. Ploidy of the obtained culture was determined. Place and Duration of Study: Department of Biotechnology, Rubber Research Institute of India and duration of the study 12 months. Methodology: Endosperm tissues were collected from Hevea seeds of different developmental stages. Somatic embryogenesis from endosperm tissue using callus mediated embryogenesis and direct method of embryogenesis were carried. Endosperm tissue from both immature and mature seeds was cut into thin slices and subjected to enzymatic digestion for the release of protoplasts. Different concentrations and combinations of cell wall digestion enzymes and osmotic agents experimented. The callus obtained from endosperm tissue was subjected to cytological analysis and flow cytometric analysis. Results: Endosperm tissue from immature fruits was found to be ideal one week between (8-10 weeks) both for somatic embryogenesis and for the release of large amount of protoplasts. Of the two basal media tried, Nitsch medium favoured callus induction, 6 % callus induction from mature endosperm tissue in presence of 2,4-D (6.3 µM) and Kin (12.1 µM) and 10 % callus induction from immature endosperm tissue in presence of  BA (4.4 µM) and NAA (2.2 µM). Direct embryogenesis (2 %) has been obtained from immature endosperm in MS basal medium along with GA3 (2.0 µM) and BA (11.1 µM). A few of the endosperm protoplasts showed division when cultured over K&M medium with NAA (0.1 µM) 2,4-D (0.2 µM) and BA (0.4 µM). Conclusion: Endosperm can be used for the development of triploids of Hevea brasiliensis. The ploidy variants i.e. triploids, developed through these in vitro techniques can be further used in Hevea brasiliensis breeding